Inappropriate responses of the immune system may cause stressful symptoms to the involved organism. Exaggerated immune answers to foreign substances or physical states which usually do not have a significant effect on the health of an animal or human may lead to allergies with symptoms ranging from mild reactions, such as skin irritations to life-threatening situations such as an anaphylactic shock or various types of vasculitis. Immune answers to endogenous antigens may cause autoimmune disorders such as systemic lupus erythematosus (SLE), type for insulin dependent diabetes mellitus (TIDM or IDDM) and different forms of arthritis.
Immune responses occur in a coordinated manner, involving several cells and requiring communication by signaling molecules such as cytokines between the cells involved. This communication may be influenced or inhibited by, e.g., interception of the signals or block of the respective receptors.
Cytokines are secreted soluble proteins, peptides and glycoproteins acting as humoral regulators at nano- to picomolar concentrations behaving like classical hormones in that they act at a systemic level and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. Cytokines differ from hormones in that they are not produced by specialized cells organized in specialized glands, i.e. there is not a single organ or cellular source for these mediators as they are expressed by virtually all cells involved in innate and adaptive immunity such as epithelial cells, macrophages, dendritic cells (DC), natural killer (NK) cells and especially by T cells, prominent among which are T helper (Th) lymphocytes.
Depending on their respective functions, cytokines may be classified into three functional categories: regulating innate immune responses, regulating adaptive immune responses and stimulating hematopoiesis. Due to their pleiotropic activities within said three categories, e.g., concerning cell activation, proliferation, differentiation, recruitment, or other physiological responses, e.g., secretion of proteins characteristic for inflammation by target cells, disturbances of the cell signaling mediated by aberrantly regulated cytokine production have been found as a cause of many disorders associated with defective immune response, for example, inflammation and cancer.
Interferons (IFN), consisting from three known protein families, type I, II and III interferons constitute one of the most important classes of cytokines. All human type I interferons bind to a cell surface receptor (IFN alpha receptor, IFN-αR) consisting of two transmembrane proteins, IFN-αR-1 and IFN-αR-2 leading to JAK-STAT activation, the formation of ISGF3 and subsequent onset of gene expression (Platanias and Fish, Exp. Hematol. (1999), 1583-1592). The composition, receptors and signaling pathways of type I IFNs have been reviewed, e.g., in Stark et al., Annu. Rev. Biochem. (1998), 227-64; Pestka S., Biopolymers (2000), 254-87. Type I interferons build a structurally related family (IFN-α (alpha), IFN-(beta), IFN-K (kappa), IFN-8 (delta), IFN-£ (epsilon), IFN-T (tau), IFN-ω (omega), and IFN-s (zeta)), of which IFN-8 and IFN-T do not occur in humans. Human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. So far, 14 IFN-α genes and 3 pseudogenes have been identified in the mouse. In humans 13 IFN-α (or IFN-α) genes (IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17 and IFN-α21) and 1 pseudogene have been identified, wherein two human IFN-α genes (IFN-α1/IFN-α1 and IFN-α13/IFN-α13) encode for identical proteins (van Pesch et al., J Viral. (2004), 8219-8228). IFN-γ is the sole Type II interferon. It is mainly involved in the induction of antimicrobial and antitumor mechanisms by macrophage stimulation. The IFN-γ receptor (IFNGR) is a heterodimeric receptor comprised of two ligand-binding IFNGRI chains associated with two signal-transducing IFNGR2 chains (Schroder et al., J Leukoc. Biol. 75 (2004), 163-189; Bach et al., Annu. Rev. Immunol. 15 (1997), 563-591). Type III interferons consist of three subtypes and are also termed IFN-α (IFN11,1 or IL-29, IFN-α2 or IL-28A and IFN-α3 or IL-28B) and have antiviral, antitumor, and immunoregulatory activity. The IFN-A receptor is also a heterodimeric complex consisting of a unique ligand-binding chain, IFN-11,R1 (also designated IL-28Ra), and an accessory chain IL-I OR2, which is shared with receptors for IL-I 0-related cytokines (Li et al., J Leukoc. Biol. 86 (2009), 23-32)
Type I interferons are pleiotropic cytokines with antiviral, antitumor and immunoregulatory functions. Depending on context, they can be anti-inflammatory and tissue protective or proinflammatory and promote autoimmunity. IFN-1a or 1b is used for the treatment of multiple sclerosis and IFN-α2b therapy for many cancers (melanoma, hemat. malig.). Elevated IFN-α activity has been frequently detected in the sera of patients with systemic lupus erythematosus (SLE) indicating that IFN-α plays a central role in SLE development (R6nnblom and Alm, J Exp. Med. (2001), F59-F63; Crow M K, Arthritis Rheum. (2003), 2396-2401; Crow M K., Curr Top Microbial. Immunol. (2007), 359-386; Crow M K. Rheum Dis Clin North Am. (2010), 173-186).
On the other side, a specific expression pattern of interferon-dependent genes (termed the “interferon signature”) is displayed in the leukocytes of patients with various autoimmune disorders such as SLE, TIDM, Sj6gren's syndrome, Dermatomyositis, Multiple Sclerosis (MS), Psoriasis and rheumatic arthritis (RA) patients. In addition, development of inflammatory arthritis, MS and TIDM has been repeatedly observed during IFN-α therapy indicating that IFN-α at least promotes those diseases (Crow M K., Arthritis Res Ther. (2010), Suppl 1:S5). Further data suggest an involvement of IFN-α in myositis, systemic scleroderma, chronic psoriasis (Higgs et al., Eur Muse Rev (2012), 22-28; Bissonnette et al., J Am Acad Dermatol (2009), 427-436; Greenberg S A, Arth Res Ther (2010): S4) and autoimmune thyroiditis (Prummel and Laurberg, Thyroid (2003), 547-551).
Accordingly, wherein depending on the context situation, treatment with Type I interferons, as in RA, MS and different leukemia or treatment with antibodies neutralizing Type I interferons, e.g., in SLE may be indicated, the very same treatment may be detrimental to the patient by promoting autoimmunity, inflammation and interferon-treatment related toxicities or even leading to the development of diseases such as MS and TIDM. One factor in these different effects may arise from the fact that, despite that different IFN subtypes activate the same cell surface receptor complex, they mediate variable responses which are also cell type dependent (van Pesch et al., J Viral 78 (2004), 8219-8228; Antonelli G., New Microbial. 31 (2008), 305-318; Gibbert et al., PLoS Pathog. 8 (2012), e1002868). The treatment therefore, should preferably be performed in a selective manner, wherein only particular IFN-α subtypes are administered to a patient, or particular IFN-α subtypes are neutralized, which are associated with a given pathological condition. Regarding IFN-α treatment such selectivity may be obtained by usage of highly purified IFN-α preparations for therapeutic purposes (Antonelli G., New Microbiol. 31 (2008), 305-318). However, regarding the use of IFN-α antibodies it is more difficult to obtain selectivity in respect of specific IFN-α subtypes, because there is a high degree of homology at the amino acid level with 80-95% homology between the IFN-α subtypes and 50% homology with IFN-β. It would be desirable therefore to provide a pool of IFN-α antibodies tolerable in humans of varying specificity towards all, selected or particular human IFN-α subtypes, which might be selectively used depending on the therapeutic and/or diagnostic indication.
First attempts to achieve this aim have already been made. For example, patent application US 2009/0214565 A1 describes isolation of several mouse anti-human IFN-α antibodies which neutralize between three and thirteen different subtypes of human IFN-α and U.S. Pat. No. 7,087,726 B2 describes a murine anti-human IFN-α antibody and its humanized version which recognize seven subtypes of human IFN-α. However, most if not all anti-IFN-antibodies provided so far are of murine origin and thus prone to an adverse reaction in humans.
Due to immunological responses to foreign antibodies, as mouse antibodies in humans (RAMA-response; Schroff et al., Cancer Res. 45 (1985), 879-885; Shawler et al., J Immunol. 135 (1985), 1530-1535), mostly humanized versions of antibodies are used in present therapeutic approaches (Chan and Carter, Nature Reviews Immunology 10 (2010), 301-316; Nelson et al., Nature Reviews Drug Discovery 9 (2010), 767-774). One approach to gain such antibodies was to transplant the complementarity determining regions (CDR) into a completely human framework, a process known as antibody humanization (Jones et al., Nature 321 (1986), 522-525). This approach is often complicated by the fact that mouse CDR do not easily transfer to a human variable domain framework, resulting in lower affinity of the humanized antibody over their parental murine antibody. Therefore, additional and elaborate mutagenesis experiments are often required, to increase the affinity of the so engineered antibodies. Another approach for achieving humanized antibodies is to immunize mice which have had their innate antibody genes replaced with human antibody genes and to isolate the antibodies produced by these animals. However, this method still requires immunization with an antigen, which is not possible with all antigens because of the toxicity of some of them. Furthermore, this method is limited to the production of transgenic mice of a specific strain.
Another method is to use libraries of human antibodies, such as phage display, as described, for example, for the generation of IL-13 specific antibodies in international application WO 2005/007699. Here, bacteriophages are engineered to display human scFv/Fab fragments on their surface by inserting a human antibody gene into the phage population. Unfortunately, there is a number of disadvantages of this method as well, including size limitation of the protein sequence for polyvalent display, the requirement of secretion of the proteins, i.e. antibody scFv/Fab fragments, from bacteria, the size limits of the library, limited number of possible antibodies produced and tested, a reduced proportion of antibodies with somatic hypermutations produced by natural immunization and that all phage-encoded proteins are fusion proteins, which may limit the activity or accessibility for the binding of some proteins. Similarly, European patent application EP O 616 640 A1 describes the production of autoantibodies from antibody segment repertoires displayed on phage. Phage libraries are generated from unimmunized humans in this respect (see, e.g., Example 1; page 16, lines 43-51; Example 2, at page 17, paragraph [0158], lines 57-58). However, also the methods described in this patent application suffer from above mentioned general disadvantages of antibodies generated from phage libraries, in comparison to antibodies produced and matured in a mammalian, i.e. human body.
The same applies to the most prominent anti-IFNa monoclonal antibody Sifalimumab (formerly, MEDI-545) that binds to and specifically neutralizes most IFN-α subtypes, preventing signaling through the type I IFN receptor. Sifalimumab is said to be a “human” anti-IFNa monoclonal antibody but actually has been derived from humanized mice, i.e. from the former company Medarex' UltiMab platform which is based on transgenic mice in which the largest fraction of the human germline repertoire was introduced. Nevertheless, though the amino acid sequences of the antibodies derived from humanized mice are of human origin these antibodies are artificial and not truly human as they have not undergone immunization, recombination, selection and affinity maturation in a human being for which reason there is still the risk of their being immunogenic and less effective, in particular compared to human-derived antibodies.
In view of the above, there is still a need for additional and new compounds like binding molecules of high specificity for particular human IFN-α subtypes, specific for a selected range or for all IFN-α subtypes which are tolerable in humans either for monotherapy or combinatorial approaches.
The solution to this problem is provided by the embodiments of the present invention as characterized in the claims and disclosed in the description and illustrated in the Examples and Figures further below.